1. Field of the Invention
This invention relates to assay methods of the homogeneous and heterogeneous specific binding type for determining qualitatively or quantitatively a ligand in a liquid medium. In a preferred embodiment, the present invention relates to competitive binding immunoassays employing nonisotopic labels.
2. Description of the Prior Art
In conventional specific binding assay techniques, the test sample is combined with reagent means of various compositions that include a conjugate of a labeling substance linked to a binding component which participates with other constituents, if any, of the reagent means to form a binding reaction system producing two species of the labeled conjugate, a bound-species and a free-species. The relative amounts of the labeled conjugate that result in the bound-species and the free-species are a function of the presence or amount of the ligand to be detected in the test sample.
As an illustration, a conventional competitive binding assay technique will now be described. In such a technique the reagent means would comprise (1) a labeled conjugate in the form of the ligand to be detected, or of an appropriate analog thereof, chemically linked to a labeling substance and (2) a specific binding partner for the ligand, such as an antibody or other binding protein. Upon combination of the test sample and the reagent means, the ligand to be detected and the labeled conjugate would compete in a substantially nondiscriminating manner for noncovalent binding to the specific binding partner. As a result, the amount of labeled conjugate that would become bound to the binding partner, i.e., in the bound-species, or that would remain free, i.e., unbound to the binding partner, and, thus, in the free-species, is a function of the amount of competing ligand present.
Where the labeled conjugate in the bound-species and that in the free-species are essentially indistinguishable by the means used to measure the labeling substance, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to as "heterogeneous." Where the bound-species and free-species forms of the labeled conjugate can be distinguished, a "homogeneous" format may be followed and the separation step avoided.
The first discovered type of specific binding assay was the radioimmunoassay which employs a radioactive isotope as the labeling substance. Such an assay necessarily must follow the heterogeneous format. Because of the hazard and difficulty of handling radioactive materials, many new assay systems have been devised using materials other than isotopes as the labeling substance, including enzymes, fluoroscent molecules, bacteriophages, coenzymes, and luminescent molecules.
The following describe several different heterogeneous binding reaction systems in which an enzyme is employed as the labeling substance: U.S. Pat. Nos. 3,654,909; 3,791,932; 3,839,153; 3,850,752; and 3,879,262; J. Immunol. Methods 1:247 (1972); and J. Immunol. 109:129(1972). A heterogeneous binding assay utilizing a non-active precursor of a spectrophotometrically detectable substance as the labeling substance is suggested in U.S. Pat. No. 3,880,934. Of further background interest pertaining to heterogeneous assays is Principles of Competitive Protein-Binding Assays, ed. Odell and Daughaday (J. B. Lippincott Co., Philadelphia, 1972). An enzyme-labeled immunoassay of the homogeneous type is described in U.S. Pat. No. 3,817,834 wherein a ligand-enzyme conjugate is employed. The enzymatic activity of the conjugate in the bound-species is measurably less than that in the free-species, thereby allowing a homogeneous format to be used. The use of particularly unique materials as labeling substances, including coenzymes, luminescent molecules, and cleavable fluorescent substrates, in both homogeneous and heterogeneous formats, is described in U.S. patent applications Ser. Nos. 667,982 and 667,996, filed on Mar. 18, 1976, and assigned to the instant assignee.
It is a primary object of the present invention to provide a specific binding assay method, useable in both the homogeneous and heterogeneous modes, which employs a unique labeling substance belonging to a large class of compounds for which there is an extensive literature background to facilitate selection of specific labels, approaches to derivatization, and monitoring reactions.